Process of isolating and separating proteolytic enzymes



0ct. 25, 1966 R. BERGKVIST 3,281,331

PROCESS OF ISOLATING AND SEPARATING PROTEOLYTIC ENZYMES Filed Oct. 19, 1962 25 Sheets-Sheet l Pier-5955 I Fo? @fo/*5455 [607- /27- p/J 4. 5;

Zur/ow?? PH 70 were 3: @ONM/mme Cet. 25, 1966 R. BERGKvlsT 3,281,331

PROCESS OF ISOLATING AND SEPARATING PROTEOLYTIC ENZYMES Filed Oct. 19, 1962 23 Sheets-Sheet 2 GELA r/NA se Aar/wry AEzoa umaawma se Aanv/ry o m 1 Emo FIGS INVENTOR Rau- .B mam/15'1- AGENT C 25; 1966 r vR. BERGKvls-r 3,281,331

PROCESS OF ISOLATING-AND SEPARATING PROTEOLYTIC ENZYMES Filed Oct. 19. 1962 25 Sheets-Sheet 4 RECUVERY l l l l l l l l j TANN/N mi/M FIG. 5

Recveky #.5 5.o 5.5 @.0 @s Pff-0 INVENTOR.

R01-F BERGKVIST Fm. 6 BY *1M w. www.

AGEA/7' Oct. 25, 1966 R. BERGKVIST PROCESS oF ISOLATING AND SEPARATING PROTEOLYTIC ENzYMEs Filed Oct. 19, 1962 25 Sheets-Sheet o 5o 60 9o 12.0 15o m0 TIME 1N MIN. INVENTOR HG 8 ROLF BERGHVIST SRA/Mn WMM AGENT Oct. 25, 1966 R. BERGKvlsT 3,281,331

PROCESS OF ISOLATING AND SEPARATING PROTEOLYTIC ENZYMES Filed Oct. 19. 1962 23 Sheets-Sheet 6 Off/CAL DENSITY RELATIVE T0 MAXIMUM ONO 1 1 260 280 300 WAVE LENGTH IN 4M FIG' q AU'IVITY RELATIVE T0 "AX/MUM I NVE NTOR.

, ROLF BERGKwsT lo H B5 www@ AEHT FIG. IOA

Oct. 25, 1966 R. BERGKVIST 3,281,331

PROCESS OF ISOLATING AND SEPARATING PROTEOLYTIC ENZYMES Filed Oct. 19, 1962 25 Sheets-Sheet 8 JuM ESTER SPLIT IN 30 MIN- J l l 1 A A I c, TAHE CONCENTRATION INVENTOR. l 1 l l l l TEMPEMGTLAE "6'. RoLF .BERGKvisT BY MI MLM,

HGENT Oct. 25, 1966 R. BERGKVIST 3,281,331

PROCESS OF ISOLATING AND SEPARATING PROTEOLYTC ENZYMES Filed Oct. 19, 1962 23 Sheets-Sheet 9 55 55 Si s??? g P 'i l l' -S u' LT.

AGENT 3 Oct. 25, 1966 R. BERGKVIST 3,281,331

PROCESS OF ISOLATING AND SEPARATING PROTEOLYTIC ENZYMES AGENT Oct. 25, 1966 R. BERGKVIST 3,281,331

PROCESS OF ISOLATING AND SEPARATING PROTEOLYTIC ENZYMES Filed Oct. 19, 1962 25 Sheets-Sheet 1l k z o z u1 i. Q n IL w o -l u s P 3 2 og' n? "OZ I .f e' Il C REMAINING Aanv/rv G S INV ENTOR. RoLF' BERG-MWST AGENT et. 25, 1966 R. BERGKvls-r 3,281,331

PROCESS oF ISOLATING AND SEPARATING PROTEOLYTIC ENZYMES Filed Oct. 19, 1962 OPA C/ TY 23 Sheets-Sheet 1 2 1 l A n L L A l 30 oo 9.o mo TIME 1N MIN- FIG. IT A OPAC/TY SOL.

n n n l l l l`t.- A l n i n l 2O ro @o ,90 loo an wo TIME IN MIN- INVENTOR FIG 7 B ROLF BERGKVIST BY MAMMMA,

AGENT' 25 1956 R. BERGKvlsT 3,281,331

PROCESS OF ISOLATING AND SEPARATING PROTEOLYTIC ENZYMES Filed Oct. 19, 1962 23 Sheets-Sheet 15 LYS/S TIME Lysls TIME IN MIN. 1N MIN.

llo

ZoD

. l A L A A 0 l l A n A o 0.2; 0.5 0. 0.5 0.6 0 0./ 0.2 0.3 0.6 6.5 0.0

Eur/ne coNcEA/TMT/oN /N :1u/mg ENZYME "ONCEVTRAT/ON "V dan( FIG. :s A F/G- 8.

oPr/ au L DEA/.s/ ry azo INVENTOR.

:so zw ROLF ERGKV/ST l l I l l l l l (2O TIME IN MIN- AGENT ct. 25, 1966 R. BERGKvlsT 3,281,331

PROCESS OF ISOLATING AND SEPARATING PROTEOLYTIC ENZYMES Filed Oct. 19, 1962 25 SheeS--SheefI 14 LYS/S T/HE /N MN- l 2. H- 5 (a ENZYME co/vcE/VTRAT/ou w en /mg- Fl G 2O RESIDLLAL Aer/wry l l h l l 2.00 300 t0n 500 600 700 800 Q00 i000 HUMAN saun INVENTOR.

ROLF BERHvsT FIG' 2l BY MAMMM,

n Gern' C@ 25 `1935 R. BERGKvxsT 3,281,331

PROCESS OF ISOLATING AND SEPARATING PROTEOLYTIC ENZYMES Filed Oct. 19, 1962 23 Sheets-Sheet l5 IWLF BERG/(WST FIG. 22 B BY MMMMMA/ 6 R. BERGKvlsT PROCESS OF ISOLATING AND SEPARATING PROTEOLYTIC ENZYMES Filed Oct. 19,

RECIPRO 3 f 5 co/vc' EN TRA TloN 1N F/G. 23A

ENZYME INHIBTION 25 Sheets-Sheet 16 INVENTOR. ROLF .B En GK vas-r uw/L MMRAMA/ AGEN-r ct. 25, 1966 R. BERGKVIST 3,231,331

PROCESS OF ISOLATING AND SEPARATING PROTEOLYTIC ENZYMES Filed Oct. 19, 1962 23 Sheets-Sheet 1'/ l l 400 2;)0 300 Ll-OO 500 y! HuMAN seRuM FIG. 7J+A INHIITION IOO I l l '00 '2.00 32:0 lfoo soo INVENTOR.

HUMAN SERUM ROLF BERGKVIST BY FIG. vr B MM-Mm AGENT Oct. 25, 1966 R. BERGKVIST 3,281,331

PROCESS OF ISOLATING AND SEPARATING PROTEOLYTIC ENZYMES Filed Oct. 19, 1962 25 Sheets-Sheet 18 INH/Blr/ON Ifo l I l J [00 200 300 #00 5 00 y@ HuMAN ssRuM FIG. 25

o INVENTOR.

30 60 9o ILO 150 lao 2/0 240 T/ME JN M/N. Rau BERGKVIST BY 91M FIG 26 #www AGENT ct. 25, 1966 R. BERGKVIST 3,281,331

PROCESS 0F ISOLATING AND SEPARATING PROTEOLYTIC ENZYMES Filed Oct. 19, 1962 23 Sheets-Shea?I 19 PROTEIN CON EN TR A T/ON FRACTION NUMBER I N VENTOR.

IG FRAeTloN NuMEfz ROL'F BERGKVI $7* FIG. 2,7 MA/Mmmm,

AGEA/T 23 Sheets-Sheet 2O R. BERG KVIST ROLF Hc-E HT .m W u.. m W B A Y BAW IHVEHTOR PROCESS OF ISOLATING AND SEPARATING PROTEOLYTIC ENZYMES Filed Oct. 19, 1962 @ct 259 w66 md @E 

1. A PROCESS OF SELECTIVELY ISOLATING AND SEPARATING PROTEOLYTIC ENZYMES, SAID PROCESS COMPRISING THE STEPS OF (A) CONTRACTING A SOLUTION OF THE PROTEASES WITH AN ION EXCHANGE AGENT SELECTED FROM THE GROUP CONSISTING OF CARBOXYL METHYL CELLULOSE AND A CARBOXY MRTHYL CROSS-LINKED DEXTRAN AT A PH BETWEEN ABOUT 2.5 AND ABOUT 8.5 AND (B) CHANGE AGENT BY PASSING THERETHOUGH AN ELUTING ELUTING THE ADSORBED POTEASES FROM SAID ION EXSOLUTION BUFFERES TO A PH-VALUE HIGHER THAN THAT AT WHICH THE RESPECTIVE POTEASES WERE ADSORBED. 